Objective To investigate the possibility of repairing articular cartilage defects with the mesenchymal stem cells(MSCs) seeded type Ⅰ collagen-glycosaminoglycan(CG) matrices after being cultured with the chondrogenic differentiation medium. Methods The adherent population of MSCs from bone marrow of10 adult dogs were expanded in number to the 3rd passage. MSCs were seeded intothe dehydrothermal treatment (DHT) crosslinked CG matrices; 2×106 cells per 9mm diameter samples were taken. Chondrogenic differentiation was achieved by the induction media for 3 weeks. Cell contractility was evaluated by the measuement of the cell-mediated contraction of the CG matrices with time inculture.The in vitro formation of the cartilage was assessed by an assayemploying immunohistochemical identification of type Ⅱ collagen and by immunohistochemistry to demonstrate smooth muscle actin （SMA）. The cells seededingCGs wereimplanted into cartilage defectsof canine knee joints. Twelve weeks after surgery, the dogs were sacrificed and results were observed. Results There was significant contraction of the MSCsseeded DHT crosslinked CG scaffolds cultured in the cartilage induction medium. After 21 days, the MSCseeded DHT crosslinked matrices were contracted to 64.4%±0.3%; histologically, the pores were found to be compressedandthe contraction coupled with the newly synthesized matrix, transforming the MSCsseeded CG matrix into a solid tissue in most areas. The type Ⅱ collagen staining was positive. The SMA staining was positive when these MSCs were seeded and the contracted CGs were implanted into the cartilage defects of the canine knee joints to repair the cartilage defects. The function of the knee joints recovered and the solid cartilaginous tissue filled the cartilage defects. Conclusion The results demonstrates that MSCs grown in the CG matrices can produce a solid cartilaginous tissuecontaining type Ⅱ collagen after being cultured with the chondrogenic differentiation medium and implanted into cartilage defects. We hypothesize that the following steps can be performed in the chondrogenic process: ①MSCs express SMA, resulting in matrix contraction, thus achieving a required cell density (allowing the cells to operate in a necessary society)； ②Cells interact to form a type Ⅱ collagencontaining extracellular matrix (and cartilaginous tissue)； ③Other factors, suchas an applied mechanical stress, may be required to form a mature cartilage with the normal architecture.
Osteoporosis is a degenerative disease characterized by decreased bone mass and destruction of bone microstructure. At present, previous studies have found that the structure and content of type Ⅰ collagen fibers are closely related to osteoporosis. However, there have been few studies on the prevention and treatment of osteoporosis using type Ⅰ collagen fibers as therapeutic targets. In this paper, the relationships between type Ⅰ collagen fibers and osteoporosis, biomechanics, bone matrix and bone strength are discussed. At the same time, the regulation of type Ⅰ collagen-related signaling pathways in osteoporosis is summarized, such as the signaling pathways of cathepsin K, transforming growth factor-β/Sma- and Mad-related protein, transforming growth factor-β/bone morphogenetic protein, c-jun N-terminal protein kinase and Wnt/β-catenin, in order to provide a new therapeutic direction for the prevention and treatment of osteoporosis.
ObjectiveTo explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. MethodsPLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. ResultsThe epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane-like layer on the scaffold. The keratin expression of the epithelium was positive. At 3 months after transplantation in situ, the vaginal mucosa showed pink and lustrous epithelialization, and the majority of scaffold degraded. After 6 months, the neovagina length was 1.2 cm, without obvious stenosis; the vaginal mucosa had similar appearance and epithelial layer to normal vagina, but it had less duplicature; there were nail-like processes in the basal layer, but the number was less than that of normal vagina. The immunohistochemistry staining for keratin was positive. ConclusionThe PLGA/collagen type I scaffolds have good cytocompatibility with the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.
Objective To analyze the contents of collagen type Ⅰ, type Ⅲ and the ratio of collagen type Ⅰ to collagen type Ⅲ in posterior rectus sheath of different person. Methods One hundred and four tissues specimen of posterior rectus sheath were obtained during patients’ abdominal operation. The contents of collagen type Ⅰand type Ⅲ were detected by using immunohistochemistry methods. The differences of collagen contents between male and female, physical work group and non-physical work group, smoking group and non-smoking group were observed. The relationships between the contents of collagen and age, body mass index (BMI), and height were analyzed, respectively. Results ① The content of collagen typeⅠand the ratio of collagen type Ⅰ／Ⅲ were both lower in male than those in female （Plt;0.01）； there were no obvious differences in the content of collagen type Ⅲ and the total amount of collagen （Pgt;0.05）. ② There were no differences between physical work group and non-physical work group with the amount and the ratio of collagens （Pgt;0.05）. ③ When compared with non-smoking group, less collagen typeⅠ(Plt;0.01) and lower ratio of collagen Ⅰ／Ⅲ （Plt;0.05） were found in smoking group； but there was no difference with content of collagen Ⅲ（Pgt;0.05）, as well as the total amount of collagen （Pgt;0.05）. ④ The total amount of collagen, the content of collagen type Ⅰand the ratio of collagen Ⅰ／Ⅲ all decreased as age increases （r=0.341, 0.392, 0.212， P＜0.001, Plt;0.05）； no obvious change was observed in the content of collagen Ⅲ （r=0.089， Pgt;0.05）. ⑤ The content and ratio of collagen had no obvious relationships with BMI and height （Pgt;0.05）. Conclusion Smoking, gender and age are all influential factors of the content and ratio of collagens in the tissue.
Objective To investigate the influence of collagen on the biomechanics strength of tissue engineering tendon. Methods All of 75 nude mice were madethe defect models of calcaneous tendons, and were divided into 5 groups randomly. Five different materials including human hair, carbon fibre (CF), polyglycolic acid (PGA), human hair and PGA, and CF and PGA with exogenous collagen were cocultured with exogenous tenocytes to construct the tissue engineering tendons.These tendons were implanted to repair defect of calcaneous tendons of right hind limb in nude mice as experimental groups, while the materials without collagenwere implanted to repair the contralateral calcaneous tendons as control groups. In the 2nd, 4th, 6th, 8th and 12th weeks after implantation, the biomechanicalcharacteristics of the tissue engineering tendon was measured, meanwhile, the changes of the biomechanics strength were observed and compared. Results From the 2nd week to the 4th week after implantation, the experimental groups were ber than the control groups in biomechanics, there was statistically significantdifference (Plt;0.05). From the 6th to 12th weeks, there was no statisticallysignificant difference between the experiment and control groups (Pgt;0.05). Positivecorrelation existed between time and intensity, there was statistically significant difference (Plt;0.05). The strength of materials was good in human hair,followed by CF, and PGA was poor. Conclusion Exogenous collagen can enhance the mechanics strength of tissue engineering tendon, and is of a certain effect on affected limb rehabilitation in early repair stages.
Objective To investigate the effects of heat injured keratinocytes (KC) supernatant on the expressions of collagen type I, collagen type III, and matrix metalloproteinase 1 (MMP-1) of dermal fibroblasts (Fb). Methods KC and Fb were isolated and cultured. Then the models of heat injured KC and Fb were reproduced in vitro, respectively. The heat injured and normal culture supernatant were collected respectively at 12 hours, and formulated as a 50% concentration of cell-conditioned medium. According to the culture medium, Fb at passage 3-5 was divided into 3 groups. Normal Fb was cultured with the conditioned medium containing 50% heat injured KC culture supernatant (group A), the conditioned medium containing 50% normal KC culture supernatant (group B), and DMEM (group C), respectively. The cells in 3 groups were collected at 24 hours. In addition， the cells in group A were collected at 0, 1, 2, 6, 12, 24, and 48 hours， respectively. Normal Fb was cultured with the conditioned medium containing 50% heat injured Fb culture supernatant. Then, the cells were collected at 0, 1, 2, 6, 12, 24, and 48 hours, respectively. The mRNA levels of the collagen type I, collagen type III, and MMP-1 of Fb were measured by real-time fluorescent quantitative PCR techniques. Results At 24 hours after cultured with supernatant of heat injured KC，mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A were significantly higher than those in groups B and C (P lt; 0.05). The mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A gradually increased with time going, showing significant differences between 0 hour and 2, 6, 12, 24, and 48 hours (P lt; 0.05); significant differences were found between different time points after 2 hours (P lt; 0.05). After Fb was treated with supernatant of heat injured Fb, the mRNA relative expression levels of MMP-1 gradually decreased with time going, showing significant differences between 0 hour and 1, 2, 6, 12, 24, and 24 hours (P lt; 0.05); after 2 hours of culture, significant differences were found among different time points (P lt; 0.05). Conclusion Heat injured KC supernatant may regulate the mRNA expressions of collagen type I, collagen type III, and MMP-1 of Fb.
Objective To investigate the effect of collagen type I concentration on the physical and chemical properties of the collagen hydrogel, and to analyze the effect of different concentrations of collagen type I hydrogel on the phenotype and gene expression of the chondrocytes in vitro. Methods Three kinds of collagen hydrogels with concentrations of 12, 8, and 6 mg/ mL (C12, C8, and C6) were prepared, respectively. The micro-structure, compressive modulus, and swelling ratio of the hydrogels were measured and analyzed. The chondrocytes at 2nd passage were cocultured with three kinds of collagen hydrogels in vitro, respectively. After 1-day culture, the samples were stained with fluorescein diacetate (FDA) / propidium iodide (PI) and the cell activity was observed under confocal laser microscope. After 14-day culture, HE staining and toluidine blue staining were carried out to observe the histological morphology, and mRNA expressions of chondrocytes related genes (collagen type II, Aggrecan, collagen type I, collagen type X, Sox9) were determined by real-time fluorescent quantitative PCR. Results With the increase of collagen type I concentration from 6 to 12 mg/mL, the physical and chemical properties of the collagen hydrogels changed significantly: the fiber network became dense; the swelling ratios of C6, C8, and C12 were 0.260 ± 0.055, 0.358 ± 0.072, and 0.539 ± 0.033 at 192 hours, respectively, showing significant differences among 3 groups (P lt; 0.05); and the compression modulus were (4.86 ± 0.96), (7.09 ± 2.33), and (11.08 ± 3.18) kPa, respectively, showing significant differences among 3 groups (P lt; 0.05). After stained with FDA/PI, most cells were stained green, and few were stained red. The histological observation results showed that the chondrocytes in C12 hydrogels aggregated obviously with b heterochromia, chondrocytes in C8 hydrogels aggregated partly with obvious heterochromia, and chondrcytes in C6 hydrogels uniformly distributed with weak heterochromia. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of collagen type II and Aggrecan were at the same level in C12, C8, and C6; the expressions of collagen type I, Sox9, and collagen type X were up-regulated with the increase of collagen type I hydrogels concentration, and the expressions were the highest at 12 mg/mL and were the lowest at 6 mg/mL, showing significant differences among 3 groups (P lt; 0.05). Conclusion Increasing the concentration of collagen hydrogels leads to better mechanical properties and higher shrink-resistance, but it may induce the up-regulation of cartilage fibrosis and hypertrophy related gene expression.
ObjectiveTo explore the interaction between immune cell infiltration and extracellular matrix (ECM) in diffuse gastric cancer (DGC), and to identify novel diagnostic biomarkers and therapeutic targets. MethodsTranscriptomic data of DGC patients from The Cancer Genome Atlas (TCGA) database were analyzed to screen potential regulator factor of immune-related and ECM receptor-related signaling pathways. Differential expression of the identified regulator was assessed between the DGC tissues and the adjacent gastric tissues. Bioinformatics analysis was utilized to evaluate the relation between the regulator factor and immune cell infiltration and ECM, as well as prognosis. The clinical validation was performed using 90 paraffin-embedded DGC tissues and adjacent gastric tissues from the patients treated at The Lanzhou University Second Hospital (hereafter “our hospital”) from January 2017 to December 2019. The immunohistochemical staining was employed to examine the expression of regulator factor, followed by analysis of its association with immune cell infiltration, clinicopathologic features, and prognosis. Additionally, 10 paired DGC tissues and adjacent gastric tissues from the patients treated in our hospital in 2024 were collected for validation using real-time quantitative PCR to assess mRNA expression. The significance level was set at α=0.05. ResultsThe collagen type I alpha 1 chain (COL1A1), a potential regulator factor linked to immune and ECM receptor signaling pathways, was identified from the TCGA database. The COL1A1 was significantly overexpressed in the DGC tissues compared to the adjacent gastric tissues (P<0.001), and its high expression correlated with poorer prognosis [HR(95%CI)=2.98(1.21, 7.30), P=0.017]. The COL1A1 gene expression negatively correlated with CD8+ T cell enrichment score (CIBERSORT: r=−0.17, P<0.001; xCELL: r=−0.32, P<0.001) but positively correlated with M2 tumor-associated macrophage enrichment score (CIBERSORT: r=0.32, P<0.001; xCELL: r=0.24, P<0.001). The clinical validation confirmed that the COL1A1 protein and mRNA were both overexpressed in the DGC tissues (P<0.001). The patients with high COL1A1 protein expression had worse overall survival (P<0.001), and high expression (vs. low) was an independent risk factor for postoperative overall survival [HR(95%CI)=6.607(3.374, 12.940), P<0.001]. The COL1A1 protein expression positively correlated with CD163 (an M2 macrophage marker; r=0.76, P<0.001) and negatively with CD8+ (T cell marker, r=−0.84, P<0.001). ConclusionThis study demonstrates that COL1A1 is a potential therapeutic target for immune suppression and ECM interaction in DGC and a critical prognostic factor for long-term survival in patients with DGC.
ObjectiveTo investigate the growth characteristics of pancreatic cancer cells in the twodimensional culture system (monolayer) and threedimensional culture system (type Ⅰ collagen and extracellular matrix gel). MethodsThree pancreatic cancer cell lines (SW1990, PCT, and ASPC1) were cultured in monolayer, type Ⅰ collagen, and extracellular matrix gel, respectively. The growth patterns were observed, growth curves were detected by CCK8 test, and the cell cycle distributions were analyzed by propidium iodide staining. Results In the twodimensional culture system, cells grew in monolayer. In the type Ⅰ collagen and the ECM gel threedimensional culture system, cells formed multicellular spheroids (MCS), of which the growth rates were slower than those of the cells in monolayer. The proportions of S phase of SW1990, PCT, and ASPC1 cells in twodimensional culture system were significantly more than those in the type Ⅰ collagen on 4 d and 8 d 〔(29.6±3.0)% vs. (18.2±5.1)%, (33.6±2.1)% vs. (14.5±3.2)%, (33.1±1.8)% vs. (24.7±2.6)%; Plt;0.05〕, while the difference of proportion of three cell lines in G2/M phase was not different between twodimensional culture system and type Ⅰ collagen (Pgt;0.05). The proportions of G0/G1 phase of SW1990 and PCT cells cultured in the type Ⅰ collagen on 4 d and 8 d and ASPC1 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in twodimensional culture system (Plt;0.05). The proportions of S phase of ASPC1 cells and SW1990 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in the type Ⅰ collagen on 8 d (Plt;0.05). ConclusionsThe characteristics of pancreatic cancer cells in twodimensional and threedimensional culture systems are different. MCS culture system can better mimic the in vivo growth environment of cells in tumors.
Objective To investigate the preventive effect of carbachol on the formation of postoperative intra-abdominal adhesion. Methods Forty-four Wistar rats were randomly divided into sham operation group (SO group, n=12), operation group (n=16) and carbachol treated group (carbachol group, n=16, carbachol 50 μg/kg). Animal model of abdominal adhesion was established by rubbing the procussus vermiformis of cecum with dry sterile gauze, and by clamping and scuffing abdominal wall. Half of rats were separately killed on day 7 and day 14 after surgery, respectively. The degree of adhesion was evaluated according to Phillips 5-scale grade and the feature of this model. The histopathological changes of adhesive tissues were observed and the content of collagen type Ⅰ in the tissues was detected by immunohistochemistry. Results The scores of intra-abdominal adhesion were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). Mild inflammatory changes and less fibrous proliferation were observed in carbachol group microscopically. The contents of collagen type Ⅰ detected by immunohistochemistry were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). There was no significant difference of the score of abdominal adhesion and content of collagen type Ⅰ in the same group between 7 d and 14 d (Pgt;0.05). Conclusion Carbachol may take a significant role in the prevention of postoperative abdominal adhesion in rat.